The invention described herein may be manufactured, licensed and used for governmental purposes without the payment of any royalties to us thereon.
The present invention relates to the use of Epstein Barr Virus glycoprotein 350/220 (Gp350/220), and naturally-occurring or synthetically-derived fragments of Gp350/220 which retain the ability to bind to the CR2 receptor on B cells. The invention also relates to non-complement derived peptides that bind to the CR2 receptor as well complement-derived peptides and the hexapeptide LYNVEA. These proteins, peptides, and fragments can be used as vaccine adjuvants and as adjuvanting components of immunostimulatory compositions and vaccines. The invention also relates to the use of non-complement derived peptides that bind to the CR2 receptors, as well as complement derived peptides and the hexapeptide LYNVEA.
Complement is the name given to a series of some 20 proteins which are activated by microbial invasion to form an important line of defense against infection. The most well-recognized complement functions are those leading to the osmotic lysis and/or phagocytosis of invading bacteria or parasites. Components in the cell walls of infectious organisms trigger the complex and interconnected pathways of the complement enzyme cascade. During this process the most abundant component, C3, is converted into an enzymatically active form and ultimately cleaved into a number of fragments such as C3a and a series of phagocytosis-promoting peptides including C3b and related peptides, iC3b, C3dg.
C3a is an anaphylatoxin which triggers mast cells and basophils to release a host of chemotactic and inflammatory factors which both contribute to the activation of neutrophils and other phagocytic cells, and concentrate these cells at the site of microbial infection. C3b becomes covalently linked to the surface of the invading organism. The bound C3b interacts with the CR1 (CD35) receptors on the surface of the phagocytic cells. This interaction induces the activated phagocytes to engulf the microbes, which are then fused with cytoplasmic granules and destroyed. The destruction of invading microorganisms by phagocytic cells is an important part of cellular immunity.
More than two decades ago, researchers found that C3 peptides can stimulate resting B cells, thus suggesting that this complement component may also play a role in the humoral immune system. Hartmann, Transplant. Rev. 23:70-104 (1975); and Hartman and Bokisch, J. Exp. Med. 142:600-610 (1975). It is now recognized that these stimulated B cells produce antibacterial antibodies that assist with the process of phagocytosis. Phagocytes are most effective in combating bacteria when the bacteria are coated with antibodies. This effect, termed opsonization, is particularly important in combating encapsulated bacteria which are generally resistant to phagocytosis. It has been suggested that the generation of opsonizing antibodies is favored by the association of bacterial surface antigens with C3 peptides. In other words, the association of C3 on the bacterial surface stimulates B cells to produce anti-bacterial antibodies. Thus, C3 not only stimulates phagocytosis directly, but also stimulates B cells to produce antibodies that bind to the invading microorganism and further promote phagocytosis.
The B cell stimulatory property of the C3 peptides does not require the entire molecule, but is contained in a short sequence containing the hexapeptide LYNVEA. Lambris et al., Proc. Natl. Acad. Sci. USA, 82:4235-39 (1985); and Frade et al., BBRC 188:833-42 (1992) (Incorporated by reference). Notably, C3 molecules, and shorter peptides containing the hexapeptide sequence are only stimulatory as multimers, indicating that cross-linking of the C3 receptor is necessary for B cell proliferation. Servis and Lambris, J. Immunol. 142:2207-12 (1989); and Tsokos et al., J. Immunol. 144:1640-45 (1990). Because many molecules of C3 can bind to a single bacterium, this condition is easily satisfied in vivo.
More recent work indicates that the immunostimulatory effect of cross-linking the C3 receptor on the B cell is mediated by lowering the activation threshold for stimulation of the antigen receptor. When the C3 receptor is cross-linked, either less antigen or antigen with a lower affinity for the antigen receptor on a B cell is required for B cell stimulation. Mongini et al., J. Immunol. 159:3782-91 (1997).
Both B cells and phagocytic cells express CR1 receptors on their cell surface. However, unlike phagocytes, B cells also express the structurally related CR2 receptors (CD21). Cross-linking of CR2 molecules on the B cell surface appears to be directly responsible for the stimulatory effect of C3d, C3dg, C3bi and iC3b peptides. Reviewed in Frade, Seminars in Immunology 2:159-64 (1990). Moreover, CR1, CR2, and another protein, CD19, appear to be associated on the B cell surface. Agents which cross-link any member of this complex result in an enhanced B cell response. This signal may be provided by multimeric C3 peptides, or by antibodies directed against one or more of these associated proteins. Nemerow et al., J. Immunol. 135:3068-73 (1985); and Kozono et al., J. Immunol. 160:1565-72 (1998); Carter and Fearson, Science 256:105-07 (1992).
Indications that crosslinking of CR2 molecules promotes B cell activation have led to the use of C3d sequences as an adjuvant. Dempsey and coworkers demonstrated that a recombinant fusion protein of hen egg lysozyme containing one copy of C3d did not appreciably change the immunogenicity of the lysozyme. However, the fusion of two or three copies of the C3d peptide increased the level of anti-lysozyme antibodies by 1000- and 10,000-fold, respectively. Dempsey et al., Science, 271:348-50 (1996).
In addition to binding complement components, the CR2 receptor has also been identified as the receptor for the B-cell lymphotropic Epstein-Barr Virus (EBV). Fingeroth et al., Proc. Natl. Acad. Sci. USA 81:4510-14 (1984); and Frade et al., Proc. Natl. Acad. Sci. USA 82:1490-93 (1985). EBV has long been recognized as a B cell mitogen and polyclonal activator of antibody synthesis. In vivo, primary EBV infection is characterized by non-specific hypergammaglobulinemia. In vitro, EBV transformed B cells secrete Ig. See review, Giovana and Blaese, Adv. Immunol. 37:99-149 (F. J. Dixon ed., 1985) (Incorporated by reference).
EBV infects over 95% of the world population and is best known as the causative agent for infectious mononucleosis. Moreover, EBV is also strongly associated with a host of pathologies including endemic Burkit""s lymphoma, undifferentiated nasopharyngeal carcinoma, X-linked proliferative disorder (XLPD), hairy cell leukemia, post-transplant lymphoproliferative disorders, and some types of Hodgkin""s lymphoma, T cell lymphomas, and gastric carcinomas. In addition, unusual EBV-derived tumors are frequently found in immunosuppressed patients, including those infected with the AIDs virus. Consequently, investigators have long sought a safe and effective vaccine to prevent EBV infection. The EBV infection process is initiated by the binding of the major EBV outer membrane glycoprotein, Gp350/220, to CR2. This interaction stimulates phagocytosis or fusion of the virus with the B cell membrane which allows the viral genome to enter the cytoplasm. Tanner et al., Cell 50:2-3-213 (1987). Interestingly, some evidence suggests that C3d and Gp350/220 bind to different sites on the CR2 receptor. Barel et al., J. Immunol. 141:1590-1595 (1988). Viral entry is via the Gp350/220 protein and most of these vaccines have focused on blocking the infection process by eliciting anti-Gp350/220 antibodies. See reviews, Morgan, Vaccine, 10:563-571 (1992); and Spring et al., J. Natl. Cancer Ctr. 88:1436-41 (1996). Of course, these vaccines are designed solely to elicit antibodies against Gp350/220.
Thus, there remains a need for safe and effective adjuvants directed at activating B cells through the CR2 receptor complex.
The present invention addresses these needs by providing vaccine adjuvants which bind to the CR2 complex. These adjuvants include an Epstein Barr Virus glycoprotein 350/220 or naturally occurring variant thereof, a fusion protein comprising EBV 350/220 sequence sufficient to bind the CR2 receptor, or a recombinant or synthetically-derived fragment of Gp350/220 which retains the ability to bind to the CR2 receptor. The adjuvants of the invention also include non-complement derived peptides that bind to the CR2 receptor as well as complement derived peptides and those related to the hexapeptide LYNVEA. Co-administration of the adjuvant with an antigen of interest, which is other than an antigen comprising EBV 350/220 sequence, enhances the immunogenicity of the antigen. In a preferred embodiment, the adjuvant is directly or indirectly covalently bound to an antigen of interest, to form an immunogenic composition. In a preferred embodiment of the composition, antibodies are elicited against at least one Gp350/220 epitope and against at least one epitope of the antigen.